The invention relates to nucleic acid constructs that comprise at least one activator sequence, at least one promoter sequence and at least one regulated gene. The as:, invention also relates to repressors that are involved in cell-cycle regulation.
One of the factors implicated in cell cycle-regulated repression is E2F, which can form DNA-binding repressor complexes through its interaction with pocket proteins, such as pRb (Weintraub et al., Nature 358, 259 (1992); Helin and Harlow, Trends Cell Biol. 3, 43 (1993); Zamanian and La Thangue, Mol. Biol. Cell 4, 389 (1993)).
E2F is a heterodimeric transcription factor composed of members of the E2F and DP multi-gene families (Nevins, Science 258, 424 (1992); Mxc3xcller, Trends Genet. 11, 173 (1995); La Thangue, Transactions 24, 54 (1996)). Another transcription factor belonging to the E2F gene family is e.g. E2F-5 (WO 96/25494). Transcriptional activation by E2F is modulated during the cell cycle by pocket proteins of the pRb family. E2F is repressed in G0 and early G1, but during cell cycle progression both DP/E2F moiety and the associated pocket proteins are hyperphosphorylated by G1-specific cyclin dependent kinases leading to the dissociation of the inhibitory ternary complex (DeCaprio et al., Proc. Natl. Acad. Sci. USA 89, 1795 (1992); Fagan et al., Cell 78, 799 (1994); Hatakeyama et al., Genes Dev. 8, 1759 (1994); Weinberg, Cell 81, 323 (1995)). This dissociation generates transcriptionally active xe2x80x9cfree E2Fxe2x80x9d and leads to the activation of E2F-regulated genes. Therefore, e.g. a vector comprising a nucleic acid encoding an E2F regulator and/or E1A regulator has been already used to transfect a differentiated neuron inducing DNA synthesis (WO 95/16774).
Among the promoters controlled by transcriptional repression through E2FBSs are E2F-1, orc-1 and B-myb (Lam and Watson, EMBO J. 12, 2705 (1993); Hsiao et al., Genes Dev. 8424, 1526 (1994); Johnson et al., Genes Dev. 8424, 1514 (1994); Ohtani et al., Mol. Cell. Biol. 16, 6977 (1996)). The role of E2F, however, is not exclusively activating. This has first been demonstrated for the mouse B-myb gene (Lam and Watson, EMBO J. 12, 2705 (1993); Lam et al., EMBO J. 13, 871 (1994); Lam et al., Gene 160, 277 (1995); Zwicker et al., Science 271, 1595 (1996)). Mutation of the E2F binding site (E2FBS) in the B-myb promoter leads to a dramatically increased activity selectively in G0 and consequently to a loss of cell cycle regulation.
Other examples in this context are the E2F, p107, histone H2A and orc1 promoters, where mutations of E2FBSs also abrogate repression and cell cycle regulation (Hsiao et al., Genes Dev. 8424, 1526 (1994); Johnson et al., Genes Dev. 8424, 1514 (1994); Zhu et al., Mol. Cell. Biol. 15, 3552 (1995); Ohtani et al., Mol. Cell. Biol. 16, 6977 (1996); Oswald et al., Mol. Cell. Biol. 16, 1889 (1996)).
The identification of several genes that are repressed through E2FBSs suggests that E2F-mediated transcriptional repression is a frequent mechanism of cell cycle regulated transcription. However, the mechanism of B-myb gene repression deviates from all models proposed for the action of E2F in that it requires a second element located directly downstream of the E2FBS (Bennet et al., Oncogene 13, 1073 (1996); Zwicker et al., Science 271, 1595 (1996)). In addition, occupation in the cell of the B-myb E2FBS is cell cycle-regulated and is seen only during phases of repression (Zwicker et al., Science 271, 1595 (1996)). These observations are very similar to those made with other promoters, such as cdc25C, cdc2 and cyclin A, which are periodically repressed through two cooperating elements, the E2FBS-like CDE and the adjacent CHR (Zwicker et al., EMBO J. 14, 4514 (1995)).
The mechanism of cell. cycle regulated transcription was discovered through the analysis of genes that are expressed at later stages of the cell cycle. When the cdc25C promoter, which is up-regulated in late S/G2, was studied by in vivo footprinting and mutational analysis, a novel repressor element, the xe2x80x9ccell cycle dependent elementxe2x80x9d (CDE), was identified (Lucibello et al., EMBO J. 14, 132 (1995)). The CDE is occupied in G0-G1 and its occupation is lost in G2, when cdc25C is expressed.
That CDE mediated repression plays a role in regulating other promoters as well was shown by the presence of functional CDEs in the cyclin A and cdc2 promoters which are derepressed in late G1/S (Zwicker et al., EMBO J. 14, 4514 (1995)). These studies also led to the discovery of an additional element contiguous with the CDE, which is identical in all three promoters. This element was termed xe2x80x9ccell cycle genes homology regionxe2x80x9d (CHR) (Zwicker et al., EMBO J. 14, 4514 (1995)).
Mutation of either the CDE or the CHR in the cdc25C, cdc2 or cyclin A promoter largely abolishes repression in G2. These functional data were supported by the demonstration of G0-G1-specific protein binding to both the CDE and CHR in genomic footprinting. Interestingly, CDE contacts the major groove of DNA while binding to CHR occurs in the minor groove (Zwicker et al., EMBO J. 14, 4514 (1995)). The nucleotide sequence of the CDE-CHR and its use for diagnosis, screening and gene therapy has already been claimed in WO 96/06943.
The discovery that the CHR cooperates with a CDE in the repression of promoters and the identification of CHR-like sequences adjacent to the E2FBS in the B-myb promoter, prompted detailed investigations into the mechanism of B-myb repression. These studies showed that the CHR-like region is indispensable for repression and acts as a co-repressor element together with the E2FBS (Bennett et al., Oncogene 13, 1073 (1996)). This region has been termed Bmyb-CHR (Bennett et al., Oncogene 13, 1073 (1996)) or DRS (Bennett et al., Oncogene 13, 1073 (1996)).
In addition, genomic footprinting clearly showed a loss of E2F site occupation paralleling the derepression of B-myb in mid-G1 (Zwicker et al., Science 271, 1595 (1996)). These observations showed that E2F-CHR sites regulate transcription of genes induced in late G1, in a similar way as CDE-CHR sites lead to derepression of genes in S or G2. In addition, these findings indicate that repressing E2F sites differ from activating E2F sites by the absence of a contiguous CHR corepressor element. Taken together, both E2F- and CDE-mediated repression, acting at different stages in the cell cycle, are dependent on promoter-specific CHR elements (Liu, N. et al., Nucleic Acids Res. 24, 2905, No. 15 (1996)).
The CDE is identical to E2FBS core sequences, such as those in the B-myb promoter (GGCGG) (Zwicker et al., EMBO J. 14, 4514 (1995)), but it remains elusive what determines the distinction of an E2FBS from a CDE. In addition, CDE or CDE-CHR binding activities have not been identified to date and the relationship of CDE binding factor(s) to the E2F family of transcription factors is unclear.
Both repressor modules repress activating sequences, located upstream of E2FBS-Bmyb-CHR or CDE-CHR. The Bmyb-CHR element inhibits an upstream activator sequence in the early phase (G0 to mid G1 phase), and the CDE-CHR to a later phase (G0 to S phase) of the cell cycle.
This finding led to the construction of genes that contain a noncell-specific, cell-specific, virus-specific and/or metabolic-specific activator sequence, cell cycle-specific promoter modules like CDE-CHR or E2FBS-Bmyb-CHR controlling the activation of the activator sequence, and a gene encoding a therapeutic protein.
Such gene constructs have been claimed for gene therapy of various diseases (see e.g. WO 96/06943; D196.05274.2; D196.17851.7; WO 96/06940; WO 96/06938; WO 96/06941; WO 96/06939). However, constructs having various characteristics are desired for optimum control of gene expression. Thus, the need remains for new nucleic acid constructs with differing cell-cycle dependent gene expression.
The inventors surprisingly found that the factors involved in repression of the B-myb regulated promoter are different from factors involved in repression of CDE-CHR regulated promoters. Upon further study, the inventors found a new binding factor that interacts with CDE-CHR regulated promoters, and identified a regulatory sequence associated with this control mechanism. From this discovery, the inventors designed nucleic acid constructs useful for controlling gene expression in a cell-cycle dependent manner.
Thus, in one embodiment, the invention relates to a nucleic acid construct comprising at least one activator sequence; at least one chimeric promoter module comprising a nucleotide sequence which binds a protein of the E2F family and binds a CDF-1 protein; and at least one gene, wherein said chimeric promoter module promotes expression of said gene.
In another embodiment, the invention relates to a process for the preparation of a three-part nucleic acid construct, said nucleic acid construct comprising at least one activator sequence; at least one chimeric promoter module comprising a nucleotide sequence which binds a protein of the E2F family and binds a CDF-1 protein; and at least one gene, said process comprising ligating the three parts together.
In yet another embodiment, the invention relates to CDF-1 protein produced by a process comprising preparing a nuclear extract from HeLa cells; and purifying this extract by affinity chromatography in the presence of an oligonucleotide comprising a CDE-CHR sequence motif.
In a further embodiment, the invention relates to a process of identifying an unknown material or molecule as containing CDF-1 inhibition or stimulation activity, comprising providing CDF-1 to a solution, in vitro or in vivo, that contains a nucleic acid construct, said construct comprising at least one activator sequence; at least one chimeric promoter module comprising a nucleotide sequence which binds a protein of the E2F family and binds a CDF-1 protein; and at least one gene, providing said unknown material or molecule to said solution; and determining the effect of adding the material to expression of said gene.